The present invention relates to an animal model for researching pulmonary tumors, and especially, a transgenic non-human animal is prepared by genetic engineering for providing a tissue-specific expression of vascular endothelial growth factor A165 (VEGF-A165) in lung cells. Therefore, it is used for establishing an animal model for investigating the therapeutic effects of variable medicines on tumorigenesis of pulmonary adenocarcinoma.
According to the statistics made by the American Cancer Society, cancers have ranked as the second one in the top 10 death cause in America. Especially, lung cancer has become the first one in the top 10 death causing cancers in the past 10 years in America. Briefly, the number of patients dead by lung cancer is about 163,500 in 2005. The number of patients having death cause of lung cancer increases from 89.5 patients in 1975 to 72 patients in 2007 per 100,000 male persons. The number of patients having death cause of lung cancer increases from 24.5 patients in 1975 to 53 patients in 2007 per 100,000 female persons. Taking a view in the whole world, the incidence rate of lung cancer among all cancers is 12.8% and the mortality rate of is up to 90%. It is more amazing that the number of persons dead by lung cancer in 1990 has increased to be 4 times of that in 1950. Till now, the incidence rate of lung cancer has been continued increasing. Thus, there is a need to analyze the induction mechanism of lung cancer in a molecular level so as to propose measures for preventing from it in future.
Lung cancer, according to the bio-characteristics and the clinical manifestation, can be divided into two gross types including small cell lung cancer and non-small cell lung cancer. The non-small cell lung cancer primarily includes pulmonary squamous cell carcinoma, pulmonary large cell lung carcinoma, and pulmonary adenocarcinoma. According to the statistics of epidemiology in Taiwanese district, among patients with lung cancers, the ratio of patients of small cell lung cancer is only 12%-15% and the ratio of patients of non-small cell lung cancer is about 85%-88%. The cells of the small cell lung cancer grow relatively fast and their metastasis probability is higher. Because their reaction rate on chemotherapy and radiation therapy is more than 80%, a systemic chemotherapy for lung cancer prevails over other therapies. On contrast, the cells of the non-small cell lung cancer grow and spread both more slowly. However, only ¼ of early staged patients with the non-small cell lung cancer, through diagnosis, are capable of having an operation and only 1/10 of patients can have an operation to excise the tumor. Further, the metastasis probability or recurring rate of the non-small cell lung cancer in a patient after such an operation is very high. In addition, most patients who are not possible to have an operation are insensible to the chemotherapy and radiation therapy (Lu and, Chang, 1991). Base on the above mentioned, the 5-year survival rate in patients with lung cancer after therapy is only 10%. Pulmonary adenocarcinoma, the most common type of the non-small cell lung cancer (40% of lung cancer) (Travis et al. 1995), induces a tumor resulted from cells classified as secretory cells including clam cell, type II alveolar epithelial cells and mucin producing cells. Pulmonary adenocarcinoma commonly occurs in the peripheral of lung (about ⅔) and the other ⅓ of it starts proliferation from the center of lung (Minna et al., 2002). Upon forming a tumor by pulmonary adenocarcinoma in a patient, it will cause distal metastasis to other organs including brain, kidney, liver, bone and so on in 80% of patients. Therefore, to investigate the mechanism related to the pulmonary adenocarcinoma inducing factors so as to propose measures for preventing from being induced thereby is more important than to cure a tumor after its formation.
The cause of lung cancer primarily consists in gene mutation induced by carcinogens contained in, for example, smoke of long-term cigar addiction or environmentally polluted air. The quantity of gene mutation accumulated for a long term of time may induce cells toward carcinogenesis and promote the growth of a tumor. In addition to cigar addiction, the long-term inflammation response in lung induced by allergens in air may cause the pulmonary adenocarcinoma. Thus, chronic diseases in the lung and family inheritance are two kinds of common cause of pulmonary adenocarcinoma. According to some reports, genetic background is another cause of lung cancers. A person having a family background with lung cancer will have a higher probability in lung carcinomagenesis. From the tests on mice, it is learned that the difference in the carcinogenic probability among mice of variable strains, under treating them with the same dosage of carcinogenic material, is obvious (Minna et al., 2002). In addition, from recent studies of the pulmonary adenocarcinoma, some common gene mutations will lead to the pulmonary adenocarcinoma. A common medicine for treating the pulmonary adenocarcinoma, for example, Geftinib (Iressa), is an anticancer medicine designed on basis of the over-expression characteristics of the epidermal growth factor receptor (EGFR) in the body of patients (Ciardiello et al., 2002; Doroshow, 2005).
The vascular endothelial growth factor (VEGF) is a type of glycoprotein commonly occurring in the form of homodimer, and seldom occurring in the form of heterodimer (Cross et al., 2003). The VEGF plays an important role in embryo development. If it performs abnormally, many diseases will be caused, for example, cardiovascular diseases, pulmonary edema, inflammation response, tumor metastasis, angiogenesis, and so on (Tammela et al., 2004). The VEGF is divided into about four major types, including A, B, C and D types, wherein the VEGF-A is found to be positively relative to the vascular permeability (Connolly et al., 1989; Becker et al., 2005) and it is able to promote the development and differentiation in organs and to promote the vascularogenesis and angiogenesis. Additionally, under normal regulation conditions, VEGF-A is related to help in wounds occlusion and to the regulation in female menstrual cycle (Ferrrara et al., 1997). A cell or a tissue under the status of hypoxia or ischemia is able to induce the expression of the hypoxia-inducible factor-1 (HIF-1) protein increasing, to promote the transcription efficiency of the VEGF-A mRNA, to promote the occurrence of the VEGF-A protein and to cause the angiogenesis. Thus, in the VEGF family, the VEGF-A is the one mostly studied by researchers and medical teams. Further, the VEGF-A is divided into four types of isoforms including VEGF-A121, VEGF-A165, VEGF-A189 and VEGF-A206. The VEGF-A165, the commonly found type of VEGF-A, primarily functions to promote the angiogenesis. In its gene transcriptant, the exon 6 is spliced and the VEGF-A165 still retains the heparin binding site, while its ability to link acetyl heparin sulfate is much lower than that of two isoforms of VEGF-A206 and VEGF-A189.
The VEGF receptor (VEGFR), a type of cell surface protein, binds with types of the VEGF for inducing an auto-phosphorylation of the receptor to promote the downstream signals delivery and cause variable physiological reactions. VEGFR is one of receptor tyrosine kinase (RTK) including three types, that is, VEGFR-1, VEGFR-2 and VEGFR-3. The VEGFR-2, also named as KDR or Flk-1, has a high affinity to VEGF-A, VEGF-C and VEGF-D and primarily functions to promote the endothelial cell's survival, hyperplasia and differentiation (Zachary, 2003). If the VEGFR-2 gene knockout vector transfers into a mouse, the mouse will die at the stage of embryogenesis. The dead mouse has a serious defect in term of vascular endothelial cell and haematopoietic precursor. Thus, it is indicated that VEGFR-2 is important to the vascular development (Shalaby et al., 1995). VEGF-A primarily binds to VEGFR-2 and the binding of VEGF-A will induce the VEGFR-2 to form a dimer so as to promote intracellular tyrosine phosphorylation, inducing a series of signals transduction. By using a synthetic drug ZD4190 competitively inhibited the combination of VEGF with VEGFR2, it will effectively block the signals transduction (Gespach et al., 2006). Base on the foregoing, the binding of VEGF-A165 and VEGFR2 is very important.
Furthermore, the expression quantity of VEGF-A165 is positively related to the growth and spread of cancer cells. An early staged cancer cells will keep proliferation and thus lead to the deficiency in both the nutrition and oxygen rendering a large amount of cells to die. Therefore, inflammation response will occur and HIF-1α will activate so as to induce a large quantity of VEGF-A165 to be secreted. The VEGF-A165 will bind the VEGFR2 and thus a downstream signal is activated to induce vasculargenesis (Gasparini, 1999; Ferrara, 2002). It is obvious that the VEGF-A165 will help the growth and metastasis in malignant tumor cells. When the cancer cells secret a large amount of VEGF-A165, vasculargenesis will be induced so as to provide sufficient nutrition and oxygen to the tumor increasing the tumor growth speed (Ferrara et al., 1997). The over-secretion of VEGF-A165 will promote degradation in extra-cellular matrix and increase the vascular permeability rendering that the tumor cells are liable to invade into the tissues (Murphy et al., 1999). Therefore, it is a popular topic to study on developments both in inhibitors and target medicines for the VEGF-A165 and related factors of the VEGF-A165.
Further, a clara cell is a non-fibrosis lung epidermal cell distributing on the epidermis respectively on bronchus and capillary bronchiole and primarily functions to protect the lung via its performance each in anti-oxidative potential and alleviating inflammation response. The clara cells are capable of secreting a large amount of so-called clara cell secretory protein (CCSP). The CCSP mRNA primarily is found on trachea, bronchus and capillary bronchiole in lung (Hay et al., 1995). Via high oxidative potential tests, it is learned that a mouse with the CCSP gene knockout has a higher level in lung injury and death rate caused by the inflammation response (Johnston et al., 1997). In addition, the CCSP can be used to estimate the marker proteins indicating the lung damage level. In a procedure of acute lung injury, the expression quantities each of the CCSP mRNA and the CCSP in the lung of a mouse treated with ozone will reduce obviously. In addition, the expression of CCSP in the pulmonary adenocarcinom cells is not detected via immunohistochemistry. Thus, in the growth procedure of a tumor, it is deemed that the CCSP functions to inhibit the growth of a tumor (Hicks et al., 2003).